Nonenzymatic glycation of albumin is a condensation reaction between glucose and reactive epsilon amino groups of lysine residues in the protein. The reaction is initiated with attachment of the aldehyde function of acyclic glucose to albumin via nucleophilic addition, forming an aldimine, also known as a Schiff base. This intermediate undergoes an Amadori rearrangement to form an amino-deoxyfructose derivative (fructosyllysine) in stable ketoamine linkage. The Amadori product may give rise to a heterogeneous group of poorly defined advanced glycation end products (AGE), the formation of which is believed to evolve through various rearrangement, dehydration, oxidation and polymerization reactions. Prior art has suggested a pathophysiologic role of AGE-modified protein in disorders associated with aging and with diabetes, and that inhibition of AGE-crosslink formation might be beneficial in the treatment of such disorders (Brownlee et al, Science 232:1629, 1986; New Engl J Med 318:1315, 1988; Vlassara, J Lab Clin Med 124:19, 1994). However, recent experimental work indicates that albumin modified by Amadori glucose adducts is an important pathogenetic factor in the development of kidney and vascular dysfunction in aging and in diabetes. Glycated albumin exists in vivo principally as the Amadori product and its concentration is driven by the ambient glucose concentration to which albumin is exposed during its residence time in the circulation. Glycated albumin normally constitutes about 1-2% of total plasma albumin, and may be increased one-and-a-half to three fold in diabetes (Cohen and Hud, J Immunol Meth 122;279, 1989).
Experimental studies have shown that Amadori-modified glycated albumin has distinct biologic effects that non-glycated albumin does not possess. These glycated albumin-induced effects, which include stimulation of matrix production by kidney and vascular cells, mimic the changes associated with glomerulosclerosis and vasculopathies, are likely mediated by ligand-receptor systems for the glucose-modified epitope in the glycated protein, and can be prevented by molecules capable of reacting with the Amadori adduct in glycated albumin (Ziyadeh and Cohen, Molec Cell Biochem 125:19, 1993; Cohen et al, Molec Cell Biochem 151:61, 1995; J Clin Invest 95:2338, 1995; Cohen and Ziyadeh, Kidney Int 45:475, 1995; Wu and Cohen, Biochem Biophys Res Comm 207:521, 1995). Such molecules may be monoclonal antibodies or other compounds which specifically bind to the fructosyllysine epitope present on glycated albumin but not present on non-glycated albumin, and which are disclosed in U.S. Pat. No. 5,223,392 and U.S. Pat. No. 5,518,720.
The deleterious biologic effects of glycated albumin make it desirable to have the means to prevent the attachment of glucose to albumin lysine-amino groups and thereby lower glycated albumin concentrations. Such means would beneficially influence the development of kidney and vascular dysfunction in aging and in diabetes by mechanisms different from those disclosed in the prior art, which are designed to neutralize the biologic effects of glycated albumin. One manner by which lowering of glycated albumin concentrations could be accomplished in people with diabetes would be with intensive regimens for control of blood glucose levels. The Diabetes Control and Complications Trial showed that reducing the concentration of glycated protein in the circulation with intensive insulin therapy lowers the risk for development of nephropathy and retinopathy (New Engl J Med 329:977, 1993). However, it is widely appreciated that implementation and maintenance of intensive regimens such as those used in the DCCT are difficult and may be risky, and that the majority of diabetic patients remain significantly hyperglycemic with current antidiabetic therapies. Further, such regimens do not apply to non-diabetic people at risk for kidney or vascular disease. Another means by which lowering of glycated albumin concentrations could be achieved is with compounds that prevent condensation of glucose with lysine amino groups. Acetylsalicylic acid (aspirin), by virtue of rapid acetylation of epsilon amino groups, can competitively inhibit this reaction (Rao and Cotlier, Biochem Biophys Res Comm 151:991, 1977; Rendell et al, J Lab Clin Med 107:286, 1986). However, the impact of widespread protein acetylation is unknown. Moreover, the glycation-inhibiting activity of aspirin is relatively weak and potential therapeutic benefits that might be ascribed to this activity are limited by the rapid hydrolysis and short half-life of acetylsalicylic acid in the blood and by side effects anticipated at doses required to inhibit glycation in vivo (Costello and Green, Arth Rheum 25:550, 1982; Rowland and Riegelman, J Phann Sci 57:1313, 1968). Other compounds which lack acetyl groups but bind to albumin in a manner that effectively interferes with the condensation of glucose with free lysine amino groups would be more desirable as glycation inhibitors.
In an in vitro experiment van Boekel et al (Biochim Biophys Acta 1120:201, 1991) reported that 2,benzene acetic acid in concentrations of 1-5 mM could reduce the amount of sugar-attached protein after incubation of commercially purchased powdered albumin with 5 mM glucose-6-phosphate. However, the concentration of the compound required to inhibit sugar attachment in vitro, and the composition and concentration of the sugar substance used to glycate the protein, do not represent in vivo conditions. Therefore, the conclusion from this study is that the compound would not be effective in inhibiting albumin glycation in vivo since the primary sugar present in the circulation is glucose, and since the concentrations of the compound required to inhibit glycation in vitro would be toxic if given to living subjects. Additionally, van Boekel et al concluded that, because the compound binds to albumin, its concentration in tissues would be too low to be of import in disease states if administered in vivo. Van Boekel et al did not perform any in vivo experiments. The van Boekel et al study does not afford any evidence that the compound could affect glycation in vivo, and contra-indicates the possibility that in vivo administration of therapeutically acceptable amounts of the compound could lower glycated albumin concentrations in living human subjects or could beneficially influence kidney or vascular dysfunction in either diabetic or non-diabetic people.
It is well known that nonenzymatic glycation under in vitro conditions does not represent that which occurs in vivo with respect to the number and nature of glycatable sites (c.f. Cohen, Diabetes and Protein Glycosylation, Springer Verlag, 1986, p.12; Diabetes and Protein Glycation, JC Press, 1996, pp. 8-9). It also is well known that although various reducing sugars such as glucose-6-phosphate can condense with protein amino groups in vitro, the concentrations required vastly exceed those found of such sugars in vivo, and that such reducing substances promote glycation that is not representative in chemistry or in nature of that which occurs in vivo. These facts lead one to conclude that the effect of diclofenac on the attachment of glucose-6-phosphate at 5 mM concentration in vitro cannot be extrapolated to the in vivo situation where glucose-6-phosphate resides intracellularly and at a lesser order of magnitude of concentration. Further, diclofenac is usually administered in daily amounts of 100-200 mg, and van Boekel et al required concentrations of 1-5 mM to achieve any inhibition of the binding of glucose-6-phosphate to albumin in vitro. The peak plasma levels of diclofenac obtainable after a dose of 100-200 mg are 1-2 ug/ml. This concentration is equivalent to 3-6 uM, which is 1000-fold less than the concentration found by van Boekel et al to be necessary to inhibit albumin glycation in vitro. These facts lead one to conclude that diclofenac would be clinically useless for inhibition of albumin glycation in vivo, since the amount required by van Boekel et al to be effective in vitro would be toxic and deleterious if administered in vivo to living subjects. Additionally, van Boekel et al emphasize that AGE, not Amadori products, are important in glycation-related disease affecting the kidneys and eyes, leading one to conclude that reducing glycated albumin per se would be without salutary effect in vivo on kidney function or in other vascular disorders. In short the available art indicates that the in vivo administration of therapeutic amounts of compounds such as diclofenac would be useless either for the purpose of lowering concentrations of glycated albumin in living subjects or for treating kidney dysfunction.
The present invention discloses the novel and unexpected discovery that therapeutically acceptable amounts of diclofenac inhibit the formation of glycated albumin in vivo and prevent the development of kidney dysfunction in living subjects.